Composition and use of phyto-percolate for treatment of disease

ABSTRACT

This invention relates generally to a method of preparation of a phyto-percolate that is derived from fresh water mixture including algae. The phyto-percolate is believed to contain an enzyme having proteolytic activity. The invention further relates to the use of the phyto-percolate in a variety of disease states.

CROSS-REFERENCE TO RELATED APPLICATION

This patent application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/741,774, filed on Dec. 2, 2005, the contents ofwhich being incorporated herein by reference in its entirety.

FIELD OF INVENTION

This invention relates generally to methods and compositions fortreating human diseases, disorders, and conditions using a preparationof a phyto-percolate isolated from a complex mixture of fresh wateralgae and other microorganisms.

BACKGROUND OF INVENTION

Enzymes have a very important use within biochemical cycles in the humanbody. The majority of acute and chronic diseases create an inflammatoryprocess that results in the destruction of surrounding tissue. Thistissue debris becomes toxic and further hinders the processes ofdetoxification, elimination and defense by way of free radicaloxidation. Proteolytic enzymes are responsible for the body'sdetoxification processes. As humans age and chronic disease processesprogress, a deficiency of the proteolytic enzymes that carry out thebody's waste detoxification processes may be experienced. This enzymaticdeficiency aids in the production of a chronic hyper-inflammatory state,and the disease process becomes much more complex.

Enzymes are the catalysts that control and direct all metabolicprocesses. Without adequate enzymes in the body, chaos reigns and theimmune system and other metabolic processes become less efficient,making tissue repair slow and poorly replicated. Proteolytic enzymes, orproteases, are enzymes capable of breaking down proteins by cleavingpeptide bonds. They are produced and utilized by every living organismon Earth for protection, nutrient breakdown and assimilation, and wasteremoval. Many degenerative diseases stem from proteolytic enzymedeficiencies, leading to the inadequate removal of carcinogenic wastesfrom the body.

It is believed that the immune system, which helps protect us fromdiseases including cancer, cardiovascular disease, and other immunedeficient or deregulated disorders, can become ineffective because ofadvanced disease state or age. Immune deficiency caused by disease stateor advancing age can impair benefits received from the use oftherapeutic drugs that may be taken for the treatment of these variousdisorders. Therapeutic drugs may lose their effectiveness in acompromised immune system as a disease state progresses due to metabolicdysfunction or poor therapeutic drug assimilation.

With advancing age, humans experience an increasing accumulation ofenvironmental influences that are believed to have toxic effects on thehuman body. An observed effect associated with aging is a less accuratetissue repair process, possibly an expression of DNA mutations caused byenvironmental factors. Because of these alterations, foreign antigens inthe way of microbes, and environmental toxins such as radiation andchemical compounds through foods, water, and air, are allowed toincreasingly invade the human organism. These environmental toxins areintroduced primarily through the mucous membranes of the intestinaltract, upper respiratory tracts and lungs.

Human genes, which are made up of double-strands of DNA, are thedirectors of tissue repair. It is believed that through advancing ageand contact with the surrounding destructive elements, the expression ofsuch DNA may become less and less accurate because of replication errorsand mutations, thus creating very different functional end products ofrepair when compared to a younger individual.

Impaired immune protection and regulation, it is believed, allows anincreasing amount of toxic environmental components to invade the cellsof our bodies. These toxic components express destructive patterns ofoxidation by way of free radical activity, thus rendering importantmetabolic processes to function inadequately. Because of biochemicalcellular destruction, dead, fractionated cellular components arecreated, adding to the toxic manifestations. White cells, which are animportant part of the immune system, congregate at the sites of tissuedestruction in an effort to slow the process down. A chemical reactionthat takes place at the site causes inflammation that further increasesthe destructive pattern. This pattern of tissue destruction, secondaryto foreign antigen invasion and the associated white cell activity, cancreate an ongoing autoimmune hyperactive inflammatory state and anincreasing amount of toxic tissue destruction and debris. Because of theincreased inefficiency of tissue repair and the ever presence ofsurrounding environmental influences, human metabolic processes becomeless and less efficient with age.

The inner lining of the blood vessels, particularly the arteries, can beaffected by this destructive pattern. Because many environmentalcontaminants are introduced into human bodies through the intestinaltract and lungs, they spread through the body by way of the vascularbed, thus coming first in contact with the inner lining of the bloodvessels. This ongoing contact in the inner lining of the arteries withtoxic free radicals results in the destructive oxidative process. Thismaintains an ongoing inflammatory state that includes cell break downand scar tissue formation in the form of sclerotic plaques. Theseplaques are made up of fibrous tissue, cholesterol, calcium deposits andnecrotic tissue (broken down cellular components). Increasing arterialrestriction and blood thickening due to pathological fibrin diminishesblood flow and alters oxygen and nutrient distribution to vital organs.This gradually increasing cellular starvation affects the functions ofthe brain, heart, kidneys, muscles, joints and other vital systems.

It is believed that accelerated DNA mutations and errors in replication,increased oxidation, inflammation, dysregulated white cell activity, andtissue destruction are the results of a gradual progression of contactwith environmental forces, including pathogenic microbials. The amountof contact depends on lifestyle and individual health care. Someillnesses either originate from excessive free radical oxidationdestruction at the body's cellular level, or cause a great increase infree radical oxidation destruction. Therefore, when the body's ownmetabolic and healing processes are unable to cope with the excess oftoxic waste products, a cycle of ongoing inflammation and disease iscreated that interferes with the body's normal immune activity andtissue repair. Tissue destruction also activates the body's coagulation,or blood-clotting, mechanism, generating a barrage of intra-vascularthrombi, or blood clots, and blood-thickening fibrin, that canprecipitate strokes, heart attacks, pulmonary emboli, kidney damage, andphlebitis.

Oxidative free radical activity becomes rampant because of the action ofthe involving white cells attempting to control the initial cause of thedestruction. The resulting pathological agents secondary to thisinfluence of white cell activity create an ongoing destructive patternupon local surrounding tissue, the endothelial cells that line thevascular bed, and the epithelial cells lining the intestinal tract. Notonly is there destructive activity upon the above-mentioned tissues butalso there is oxidative breakdown or pathological activation of thecoagulation factors. This includes pathologically activated fibrinogento produce a soluble fibrin that, unlike insoluble fibrin, which is animportant component of the normal blood-clotting mechanism, cannot becross-linked and is pathological, or harmful to the body. This solublefibrin not only negatively influences general capillary circulation butalso kidney filtration, oxygen exchange within the alveoli of the lungs,and oxygenation of brain tissue. It not only thickens the blood, but isin itself an oxidative free radical, and contributes to the degenerativeoxidation process.

Much of the expressed symptomatology from the production of solublefibrin is caused by gram-negative bacteria, mycoplasma and Candidaalbicans, which are allowed to flourish in the immune-compromisedenvironment created by excess wastes and fibrin, and is related to thecellular destruction and by-products of ongoing free radical activity.Fibrinolytic activity, or the process of breaking down fibrin, alongwith the eradication of the foreign pathological agents by othertherapeutic interventions, can lead to increasingly effective immunesystem and white cell activity, and will greatly accelerate the healingprocess.

Most cancer processes liberate hydrogen peroxide, which acts as a freeradical oxidative agent. In addition to hydrogen peroxide, the effectsof cancer growth and chemotherapy produce excess soluble fibrin productsas a response to these abnormal and destructive processes. The fibrin isproduced as part of the body's natural reaction to tissue damage, whichalso occurs normally at the site of a superficial wound. However, at thesite of cancer growth, fibrin coats cancer cells, tragically insulatingthem from destruction by the body's immune system. These coagulationmechanisms, stimulated by the oxidative damage associated with chronicillness, the damaging effects of chemotherapy, and the nature ofabnormal cancer growth, all lead to further damage. Chronic illnessessuch as cancer produce an acceleration of disseminated intravascularcoagulation, causing not only a build-up of soluble fibrin but also ofsmall intravascular thrombi, or clots that float around the vascular bedacting as emboli that obstruct circulation. The use of a fibrinolyticagent, along with any other therapeutic regime, will increase immuneregulation and the effectiveness of white cell activity, improvecapillary circulation and nutrient flow to the body's organs, aid ineliminating toxins, and enhance the benefits of other therapeuticagents. In addition, fibrinolytic agents will reduce the amount of freeradical soluble fibrin that accelerates degenerative oxidation, and canincrease the body's immune effectiveness in combating cancer growth.

In vivo laboratory monitoring of disease processes has supported theobservations that improved cellular function and efficiency come withless oxidative, free radical activity, improved cellular nutrition,enhanced immune activity and white cell function and improvedoxygenation.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for treating orpreventing a disorder in a mammal (e.g., human, dog, cat, horse, etc.)by administering to the mammal a therapeutically effective amount ofphyto-percolate or derivative thereof.

In useful embodiments, the phyto-percolate derivative is a proteinhaving a molecular weight of about 67.5 kDa, a protein having amolecular weight of about 21.0 kDa, or a polysaccharide. In anotherembodiment, the phyto-percolate derivative has fibrinolytic enzymaticactivity. The phyto-percolate derivative may be isolated from thephyto-percolate or it may be produced by any appropriate method known inthe art. Suitable methods for producing the phyto-percolate derivativeinclude, for example, recombinantly expressing the derivative (e.g.,protein) by a microorganism and synthetically producing a derivative(i.e., chemical (cell-free) synthesis). The recombinant microorganismmay be one or more of the species present in ATCC Deposit #PTA-5863, orit may be any other appropriate specie.

In particular embodiments a particular dosage is between about 1 andabout 8 ounces per day of the phyto-percolate. Particularly noted is adosage of about 1 to about 4 ounces per day. Preferably, thephyto-percolate that is administered to the human contains between about10 ppm and about 150 ppm of a phyto-percolate derivative. In anotheruseful embodiment, a therapeutically effective amount of one or more ofthe derivatives is administered to the human. Preferably, the mammal isadministered between about 1 mg and 1000 mg of the derivative per day.Suitable methods for administration of the phyto-percolate include oraladministration. Suitable methods for administration of a phyto-percolatederivative (e.g., an isolated derivative) include, for example, oral,topical, rectal, or vaginal administration as well as intravenous,intramuscular, and subcutaneous injection.

Another aspect of this invention is directed to a method of treating anoverweight condition or obesity comprising administering to the mammal(e.g., human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingtype I and II diabetes comprising administering to the mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treating aninflammatory disorder comprising administering to the mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof. It is believed that the phyto-percolate andderivatives have broad spectrum anti-inflammatory properties andtherefore may be used to reduce or prevent inflammation in a wide rangeof diseases and disorders.

Another aspect of this invention is directed to a method for treating astomach disorder comprising administering to the mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof. Stomach disorders amenable to treatment with thephyto-percolate and/or derivatives thereof include, for example, astomach ulcer and gastric reflux disease.

In another aspect of this invention, the phyto-percolate or derivativesmay be used to alleviate side-effects of another primary therapy. Forexample, the phyto-percolate may be administered to reduce the oxidativestress, chemotherapy-induced nausea, chemotherapy-induced liver damage,appetite suppression, hair loss, fingernail and toenail loss anddiscoloration that result from anti-AIDS therapy and anti-cancer therapy(e.g., chemotherapy and radiation therapy).

In another aspect of this invention, the phyto-percolate or derivativesmay be used to reduce the recovery time in mammals (e.g., humans andhorses) after periods of stress (e.g., exercise). In a related aspect,the phyto-percolate or derivatives are administered in order to restorephysical energy and mental acuity following periods of stress.

In another aspect of this invention, the phyto-percolate or derivatesmay also be administered topically directly to the eye (e.g., in theform of eye drops) to treat lesions of the cornea, dry eyes, and similarocular disorders.

Another aspect of this invention is directed to a method for treatingconditions or disorders associated with infectious disease (e.g., aviral infection) comprising administering to the mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof. Infectious disease may be the cause of many of the above andbelow listed diseases such as pneumonia, all viruses, acariosis, acne,adenovirus, AIDS, amebiasis, anthrax, athlete's food, babesiosis,bartonellosis, Bell's palsy, botulism, candidiasis, carbuncles, Chaga'sdisease, chicken pox, Chlamydia, coccidiomycosis, coronavirus,cryptococcosis, cytomegalovirus, Dengue fever, echovirus, erysipelas,furuncle, gangrene, Guillan-Barre syndrome, hepatitis, impetigo,influenza, leucopenia, Lyme's disease, malaria, martolditis, measles,mumps, mycobacterium, mycosis, parasites, pediculosis, P.I.D. pyodermia,rabies, rubella, salmonella, salpingitis, septicemia, shingles,sinusitis, syphilis, tetanus, Tindi Cruzi and warts.

Another aspect of this invention is directed to a method for treatingdiseases related to the heart, blood vessels, renal, liver, andendocrine system comprising administering a therapeutically effectiveamount of a phyto-percolate or derivative thereof.

Another aspect of this invention is directed to a method for treating avasospasm comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingheart failure comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingcardiac hypertrophy comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingdysregulated blood pressure comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingangina comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingperipheral vascular disease comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingcerebral diseases and diseases related to the central nervous systemthat are vascular in origin comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingneuro-degeneration comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingAlzheimer's disease comprising administering to a mammal (e.g., human) atherapeutically effective amount of a compound of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingdepression comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingaddiction, including drug detoxification and/or substance abuseincluding nicotine, cocaine and alcohol abuse comprising administeringto a mammal (e.g., human) a therapeutically effective amount of aphyto-percolate or derivative thereof.

Another aspect of this invention is directed to a method for treatingattention deficit disorder and attention deficit hyperactivity disordercomprising administering to a mammal (e.g., human) a therapeuticallyeffective amount of a compound of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingsleep disorders comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingseasonal affective disorder comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingenvironmental and food allergies comprising administering to a mammal(e.g., human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingconditions related to pain or nocioception comprising administering to amammal (e.g., human) a therapeutically effective amount of aphyto-percolate or derivative thereof.

Another aspect of this invention is directed to a method for treatingmigraine comprising administering to a mammal (e.g., human) atherapeutically effective amount of a compound of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingdisorders related to disruption of circadian rhythms including jet lagcomprising administering to a mammal (e.g., human) a therapeuticallyeffective amount of a phyto-percolate or derivative thereof.

Another aspect of this invention is directed to a method for treatingdiseases related to abnormal gastrointestinal motility, secretion,and/or function comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingdiarrhea and/or incontinence comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treating agastric ulcer comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingirritable bowel syndrome comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatinginflammatory bowel disease comprising administering to a mammal (e.g.,human) a therapeutically effective amount of a phyto-percolate orderivative thereof.

Another aspect of this invention is directed to a method for treatingnausea comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingsexual dysfunction comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for alteringfertility comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate or derivativethereof.

Another aspect of this invention is directed to a method for treatingconditions or disorders associated with the immune system comprisingadministering to a mammal (e.g., human) a therapeutically effectiveamount of a phyto-percolate. Immune system deficiency may be the causeof many of the above and below listed diseases such as cancer,emphysema, encephalitis, environmental sensitivity, erysipelas, foodpoisoning and Reynaud's disease.

Another aspect of this invention is directed to a method for treatingconditions or disorders associated with hormonal imbalances comprisingadministering to a mammal (e.g., human) a therapeutically effectiveamount of a phyto-percolate. Hormonal imbalances may be the cause ofmany of the above and below listed diseases such as acne, Addison'sdisease, endometriosis, Grave's disease, osteoporosis, menstrual andmenopausal regulation, glucose, and other metabolic regulation. In thisregards, the phyto-percolate and derivatives may be used to improve thegeneral health and overall function of metabolic organs like the kidney,liver, and pancreas. It is believed that the phyto-percolate andderivatives improve the efficiency of those organs and increases theirmetabolic and endocrine functions.

Another aspect of this invention is directed to a method for treatingconditions or disorders associated with neurological deficienciescomprising administering to a mammal (e.g., human) a therapeuticallyeffective amount of a phyto-percolate. Neurological deficiencies may bethe cause of many of the above and below listed diseases such as LouGehrig's disease, chronic pain, Huntingdon's Chorea, diabeticneuropathy, multiple sclerosis, Myasthenia Gravis, Parkinson's disease,poliomyelitis, senile dementia, nigrostriatal degeneration, stroke,tardive dyskinesia and tinnitus.

Another aspect of this invention is directed to a method for treatingrespiratory diseases comprising administering to a mammal (e.g., human)a therapeutically effective amount of a phyto-percolate.

Another aspect of this invention is directed to a method for treatingasthma comprising administering to a mammal (e.g., human) atherapeutically effective amount of a phyto-percolate.

Another aspect of this invention is directed to a method for treatingdiseases related to abnormal hormone release and utilization comprisingadministering to a mammal (e.g., human) a therapeutically effectiveamount of a phyto-percolate.

Another aspect of this invention is directed to a method for treatingabnormal insulin release and utilization comprising administering to amammal (e.g., human) a therapeutically effective amount of a compound ofa phyto-percolate.

Another aspect of this invention is directed to a method for treatingskin lesions and disorders.

In addition to the “direct” effect of the phyto-percolate of thisinvention there are diseases/conditions wherein subjects with saiddiseases/conditions will benefit from the associated weight loss, andmetabolic and immune system regulation, such as insulin resistance withimpaired glucose tolerance, Type II Diabetes, hypertension,hyperlipidemia, cardiovascular disease, gall stones, certain cancers,sleep apnea, etc. resulting from use of phyto-percolate.

In a further illustrative embodiment a method of making the inventivephyto-percolate is disclosed. The phyto-percolate is prepared bycultivating a mixture of freshwater algae and bacteria that is augmentedby a nutrient blend that is related to the production of fibrinolyticenzymes, proteins, and other molecules, forming a fortified algaeculture. Added to this fortified algal and bacterial culture is purifiedfresh water that has been purified by reverse osmosis, distillationand/or deionization. The culture is percolated with said purified freshwater and nutrient blend for a predetermined time forming aphyto-percolate that is fibrinolytic and proteinaceous in nature. Thephyto-percolate is decanted from the fortified algal and bacterialculture and processed. Suitable methods of processing thephyto-percolate include filtration, centrifugation, lyophilization,purification, dilution, and other methods. The filtering of the decantedphyto-percolate in one particular embodiment is by micro-filtrationwhere the micro-filtration removes particles larger than about 0.22 μm.

In another aspect, this invention provides a substantially pure compoundisolated from a phyto-percolate. In a preferred embodiment, the compoundis isolated from the percolate produced by culturing the microorganismsof ATCC Deposit #PTA-5863 or other appropriate species as describedherein. In another embodiment, the compound is a protein having amolecular weight of about 67.5 kDa.

In a related aspect, the invention provides a pharmaceutical formulationcomprising a substantially pure compound isolated from a phyto-percolateand a pharmaceutically acceptable excipient.

The term “inflammatory disorder” encompasses a variety of conditionsincluding conditions related to a hyperactive immune system, chronicinflammation, and autoimmune disorders. Inflammatory disorders include,for example, acne vulgaris; acute febrile neutrophilic dermatosis; acuterespiratory distress syndrome; Addison's disease; adrenocorticalinsufficiency; adrenogenital ayndrome; allergic conjunctivitis; allergicrhinitis; allergic intraocular inflammatory diseases, ANCA-associatedsmall-vessel vasculitis; angioedema; ankylosing spondylitis; aphthousstomatitis; arthritis, asthma; atherosclerosis; atopic dermatitis;autoimmune disease; autoimmune hemolytic anemia; autoimmune hepatitis;Behcet's disease; Bell's palsy; berylliosis; balanitis circumscriptaplasmacellularis; balanoposthitis; bronchial asthma; bullousherpetiformis dermatitis; bullous pemphigoid; carditis; celiac disease;cerebral ischaemia; chronic obstructive pulmonary disease; cirrhosis;Cogan's syndrome; contact dermatitis; COPD; Crohn's disease; Cushing'ssyndrome; dermatomyositis; diabetes mellitus; discoid lupuserythematosus; eczema (e.g., asteatotic eczema, dyshidrotic eczema,vesicular palmoplantar eczema); eosinophilic fascitis; epicondylitis;erythema annulare centrifugum; erythema dyschromicum perstans; erythemamultiforme; erythema nodosum; exfoliative dermatitis; fibromyalgia;focal glomeruloscierosis; giant cell arteritis; gout; gouty arthritis;graft-versus-host disease; granuloma annulare; hand eczema;Henoch-Schonlein purpura; herpes gestationis; hirsutism;hypersensitivity drug reactions; idiopathic cerato-scleritis; idiopathicpulmonary fibrosis; idiopathic thrombocytopenic purpura; inflamedprostate; inflammatory bowel or gastrointestinal disorders, inflammatorydermatoses; juvenile rheumatoid arthritis; laryngeal edema; lichennitidus; lichen planus; lichen sclerosus et atrophicus; lichen simplexchronicus; lichen spinulosus; Loeffler's syndrome; lupus nephritis;lupus vulgaris; lymphomatous tracheobronchitis; macular edema; multiplesclerosis; musculoskeletal and connective tissue disorder; myastheniagravis; myositis; nummular dermatitis; obstructive pulmonary disease;ocular inflammation; organ transplant rejection; osteoarthritis;pancreatitis; pemphigoid gestationis; pemphigus vulgaris; polyarteritisnodosa; polymyalgia rheumatica; primary adrenocortical insufficiency;primary billiary cirrhosis; pruritus scroti; pruritis/inflammation,psoriasis; psoriatic arthritis; Reiter's disease; relapsingpolychondritis; pyoderma gangrenosum; rheumatic carditis; rheumaticfever; rheumatoid arthritis; rosacea caused by sarcoidosis; rosaceacaused by scleroderma; rosacea caused by Sweet's syndrome; rosaceacaused by systemic lupus erythematosus; rosacea caused by urticaria;rosacea caused by zoster-associated pain; sarcoidosis; scleroderma;segmental glomerulosclerosis; septic shock syndrome; serum sickness;shoulder tendinitis or bursitis; Sjogren's syndrome; Still's disease;stroke-induced brain cell death; Sweet's disease; systemicdermatomyositis; systemic lupus erythematosus; systemic sclerosis;Takayasu's arteritis; temporal arteritis; thyroiditis; toxic epidermalnecrolysis; tuberculosis; type-I diabetes; ulcerative colitis; uveitis;vasculitis; and Wegener's granulomatosis.

The term “substantially pure,” when referring to a protein or otherderivative of the phyto-percolate, means the state of a substance thathas been separated from the other components of the phyto-percolate.Typically, a substantially pure derivative is at least 80%, by weight,free from the proteins and other organic molecules of thephyto-percolate. Preferably, the substantially pure derivative is atleast 90%, 95%, or 99%, by weight, free from those organic molecules. Asubstantially pure protein derivative may be obtained, for example, byextracting it from a source other than the phyto-percolate. A proteinderivative, for example, may be recombinantly expressed in anothermicroorganism or in a cell-free translation system.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments, taken in conjunction with the accompanyingdrawing in which:

FIG. 1 is a flow chart showing a method of preparing a phyto-percolate;

FIG. 2 is an HPLC chromatogram of the diluted phyto-percolate;

FIG. 3 is an FTIR spectrum of the diluted phyto-percolate; and

FIG. 4 is a [¹H]-NMR spectrum of the diluted phyto-percolate.

DETAILED DESCRIPTION

The present invention provides a phyto-percolate that has therapeuticand other beneficial properties when administered to humans and otheranimals. Without being bound by any theory, it is believed that at leastone of the therapeutically active agents in the phyto-percolate is anenzyme. Methods for preparing the phyto-percolate are also provided.Detailed embodiments of the present invention are disclosed herein,however, it is to be understood that the disclosed embodiments aremerely exemplary of the invention, which may be embodied in variousforms. Therefore, specific functional details disclosed herein are notto be interpreted as limiting, but merely as a basis for the claims andas a representative basis for teaching one skilled in the art tovariously employ the present invention in virtually any appropriatelydetailed embodiment.

Phyto-Percolate Production

According to the invention, a phyto-percolate is derived from a culturecomprised of freshwater algae, moss, bacteria, actinomycetes, and fungi.It is believed that the culture is comprised of at least one or more ofthe following genera: Acinetobacter Aerococcus Aquaspirillium BacillusBrevibacterium Caseobacter Chlorella Clavibacter CorynebacteriumDermacoccus Liefsonia Micrococcus Oedocladium PhyllobacteriumPseudomonas Ralstonia Rhizobium Rhodococcus Riemerella ShingomonasStaphylococcus Stenotrophomonas Stichococcus Streptomyces UlothrixVariovorax Weeksella Xanthomonas

Particular note is made of the genera Aquaspirillum, Bacillus,Pseudomonas, Ralstonia, Stenotrophomonas, Stichococcus, and Ulothrix.Without being bound by any theory, it is believed that these genera arethe most abundant organism in each culture and may be the primaryprocuders of the phyto-percolate derivatives. A deposit of a cultureresulting in a phyto-percolate of the present invention has been placedin the American Type Culture Collection, of Manassas, Va., as Deposit #:PTA-5863.

In particular embodiments, a heterotrophic rotifer species exists in thecultures, as well as bacteria that have been identified asStenotrophomonas maltophilia, Ralstonia pickettii, Ralstonia paucula,Acinetobacter genospecies 11, Acinetobacter junii, Leifsonia aquatica,Riemerella anatipestifer, Variovorax paradoxus, and Streptomycesgriseorubens. Without being bound to any particular theory, it isbelieved that these bacteria may produce proteolytic enzymes that arecontributors to the effectiveness of the phyto-percolate.

A method of producing phyto-percolate is depicted in FIG. 1.Phyto-percolate cultures of approximately 100-200 ml of dense algalcells in approximately 2.5 gal, or approximately 10 liters, ofreverse-osmosis purified sterile water are fed about 1 milliliter (ml)per week of liquid extract of live active yeast, or Baker's yeast,Saccharomyces cerevisiae, which has been prepared from 1.0 g dry activeyeast added to 50 ml warm water, at between about 37° and about 43° C.The mixture is allowed to incubate for 10-30 minutes, or until itslightly foams. The cultures are fed in either 1.0 ml weekly doses, or0.5 ml twice-weekly doses. It is contemplated within the scope of theinvention that other yeast cultures may be used. It is furthercontemplated that other organic nutrients or substrates known in the artmay be used such as glucose or proteose, or other algal growth mediaprepared from inorganic nutrients, supplements, and/or vitamins.

In one embodiment, the cultures are grown under full-spectrum growlights at about 25° C., and produce a final unadjusted pH of betweenabout 6.2 to about 7 that fluctuates. The cultures are grown in clearglass fishbowl containers having a volume of approximately 2.5 gal withsemi-transparent plastic lids, with the exception of about a 3 mm holein the lid for gas exchange. It is contemplated within the scope of theinvention that other culture containers, ingredients, conditions andmethods known in the art may be used that allow the cells to grow in amanner in which the phyto-percolate derivatives are expressed. Suchmethods may include larger batch, semi-continuous, continuous or othertype culture systems including bireactors or photoreactors, may or maynot include aeration or agitation, may or may not include solid, liquid,semi-solid or other form of growth media or substrate, may or may notinclude the above particular conditions of temperature, contact time orarea, or light intensity.

In this particular embodiment the cultures are harvested weekly orbi-weekly, between the 5^(rd) and 10^(th) day after feeding, by drawingoff the top 1.25 gal of phyto-percolate from each 2.5 gal culture. Thisis referred to as the “raw phyto-percolate.” The algal or other cellsand yeast food forming the phyto-percolate culture remain in the bottomof the culture container substantially undisturbed while thephyto-percolate is decanted. The decanted material is then processed asdesired. The volume of the container is then optionally returned tooriginal volume. Conveniently this is accomplished with reverse-osmosispurified water at approximately room temperature, about 25° C. It iscontemplated within the scope of the invention that other culture andharvest systems, timetables volumes and methods may be used that resultin phyto-percolate derivatives.

Without being bound by any particular theory, it is believed thepatterns of harvest and feeding affect enzyme production. It is believedthat more frequent smaller feedings such as 0.5 ml twice-weekly maystimulate greater enzyme production than single large amount feedingssuch as 2 ml bi-weekly, while discouraging contamination withundesirable bacteria and rotifer colonization. Since enzyme systems arehighly dynamic and are directly affected by the immediate surroundings,the suggestion is supported that a food blend such as a liquid extractof active Baker's yeast increases the active proteolytic enzymes in thephyto-percolate culture compared with other foods or nutrient blends.

The peaks of enzyme concentration in the percolate over the course ofseveral weeks are mapped under various feeding regimens, and serve todictate the optimal date for harvests. According to the invention, theenzyme concentration is analyzed in the cultures and processedphyto-percolate to detect any negative effects of regular harvesting onthe algal cultures over time, and is combined with data on the effectsof environmental and stress factors such as dark/light, starvation,and/or changes in temperature or pH, which may stimulate or discourageenzyme production. Methods for analyzing these parameters include theisolation and homogenization of select cultures to eliminate allvariables besides those being tested, and include monitoring ofchlorophyll, total protein and enzyme activity, utilizingspectro-photometric methods, to measure the health and enzyme activityof the cultures over the course of an isolated-variable experiment.

In this particular embodiment the method for analyzing proteolyticactivity is a typical chromogenic assay using Chromogenix substrate fromDiaPharma, S-2251: chromogenic substrate for plasmin andstreptokinase-activated plasminogen. Chromogenic substrates are peptidesthat react with proteolytic enzymes and proportionally change color asthe substrate is lysed by the enzymes. The color change may be measuredspectro-photometrically over time and is proportional to the proteolyticactivity. The synthetic chromogenic assay substrates are designed tohave enzyme binding selectivity similar to that of the enzyme's naturalsubstrate. It is believed that the enzymes present in phyto-percolateare selective for substrates including fractionated proteins and fibrin.It is contemplated within the scope of the invention that other methodsfor analyzing proteolytic activity and phyto-percolate derivatives maybe used.

Enzyme activity for samples of described phyto-percolate currentlyranges from 15-50 mU/mL of plasmin-like activity, when phyto-percolateis prepared as described. These values have been found to correlate withclinical observations of reduced pathological fibrin in humans orallyconsuming phyto-percolate. Methods for evaluating in vivo effects ofphyto-percolate include peripheral blood observations on wet and dryblood smears, diagnostic and/or analytical blood tests, and variousclinical observations and measurements such as body weight. Reductionsin excess pathological fibrin and platelet aggregation have beenobserved, which are secondary to inflammation and tissue destruction.Changes in white blood cell mobility and number have also been observed.Anti-inflammatory effects of phyto-percolate in vivo have also beenmonitored with independent blood laboratory studies focusing on chronicinflammatory activity and hyper-coagulant states.

In an alternative embodiment, the phyto-percolate may be produced usinga continuous culture format in which the phyto-percolate issubstantially continuously removed from the culture and the lost volumeis replaced with fresh culture media and/or nutrients. Further, thephyto-percolate may be produced using a bioreactor that is suitable forproduction on a larger scale than the batch culture method describedabove.

Phyto-Percolate Filtration

After harvest of the phyto-percolate from the cultures, the decantedfluid is filtered through a series of depth prefilters and sterilemembrane filters made of low-protein binding materials. Examples ofsuitable final sterilizing filters are provided by Millipore Corp.Durapore brand filters, made of PVDF material. These have been shown toprotect the enzyme concentration, and provide a final sterile filtrationlevel of about 0.22 microns, as well as being chemically inert toozonated water. Ozonated water is used for sterilizing the filtersystem, as it does not leave a damaging residue like chlorine.

All filters are 10″ cartridge membrane or depth filters of variouschemically-inert materials. The prefilters are housed in cartridgefilter housings made of styrene-acrylonitrile (SAN). The final filtersare housed in polypropylene (PP) housings with Kynar fittings. Thematerial is harvested and filtered using Tygon tubing, peristaltic pumpsand 55 gallon containers or other containers that have beenpre-sterilized with ozonated water.

The phyto-percolate passes through a filtration regimen comprised of twopre-filters in SAN housings of pore size 1 μm (nominal), made of pleatedcellulose/polyester. Examples of these filters are manufactured byCole-Panner, Vernon Hills, Ill., USA, catalog number EW-29830-20. It iscontemplated within the scope of the invention that other filters knowin the art may be used in this step as pre-filters, that are chemicallyinert.

The phyto-percolate is again filtered using a second stage pre-filtermade of polypropylene in a polypropylene housing, with a nominal poresize of about 0.5 um. In one illustrative embodiment, this finishingfilter is manufactured by Millipore Corporation, Bedford, Mass.,Durapore® brand, Catalog # D00501S01. It is contemplated within thescope of the invention that other filters known in the art may be usedin this step as second pre-filters, that are chemically inert.

The phyto-percolate is then passed through a pre-sterilized final filterthat sterile-filters the phyto-percolate and removes all traces ofbacteria, yeast, mold, algae and other particle contaminants. Accordingto the invention, a final filter set consists of sterile membranefilters in PP housing having progressively smaller pore sizes of 0.45 umand 0.22 μm (absolute). These finishing filters' membranes are made ofhydrophilic extremely-low protein-binding PVDF. In one illustrativeembodiment, these finishing filters are manufactured by MilliporeCorporation, Durapore® brand, Catalog #'s CVHI01TPE and CVDI01TPE. It iscontemplated within the scope of the invention that other filters knowin the art may be used that are inert to the phyto-percolate derivativesand processing and sanitizing materials including ozonated water. It isalso contemplated within the scope of the invention that other methodsof processing may be used.

Filtration by size exclusion removes approximately >99.9% ofcontaminants such as bacteria, yeast and mold spores, and algal cells.It is also believed to preserve enzymatic activity if filter materialsare made of low-protein-binding, chemically-inert materials. Theresulting liquid, the phyto-percolate, is substantially comprised ofwater, active enzymes, proteins and sugars. The phyto-percolate, afterpassing through the finishing filter is then usefully stored in sealedsterile 55 gal HDPE drums at between 21° and 27° C. until bottling.Samples are taken from each batch immediately after filtering to testfor enzyme efficacy and contamination and for standardization. It iscontemplated within the scope of the invention that other methods ofsampling and testing may be used. The acceptable values for fibrinolyticenzyme efficacy to be administered p.o. are observed in thephyto-percolate as between 0 and 50 milli-units of plasmin-likeactivity; however higher levels may provide greater therapeutic benefit.It is believed that this filtered phyto-percolate contains approximately50 ppm of the 67.5 kDa protein (see below).

The phyto-percolate is processed and bottled under sanitary conditionsknown in the art using ozone sterilization. It is believed that thisstep avoids enzyme degradation associated with the use of chlorine orheat sterilization because ozone leaves no residue if left to dissipate,or if followed by a rinse of sterile water. It is contemplated withinthe scope of the invention that other methods of filtration andsanitization known in the art may be used that are not unreasonablydegrading of the enzymatic or other activity. The phyto-percolate isusefully packaged in opaque UV-protectant bottles and shipped with coldpacks to reduce product degradation. It is contemplated within the scopeof the invention that other methods of packing, bottling, storing, andtransporting may be used.

Phyto-Percolate Characterization

It is believed that the raw phyto-percolate, prior to filtration, is acomplex mixture of macromolecules. It was expected that the filtrationprocess described above reduced the molecular complexity of thephyto-percolate filtrate. We performed several physico-chemical tests todetermine the composition of the filtrate. In each case, thephyto-percolate filtrate was lyophilized, redissolved in ddH₂O, andrefiltered to remove any undissolved particulate matter.

A sample of the lyophilized phyto-percolate was subjected to isocraticreverse phase HPLC, on a size-exclusion chromatography column (TSK-GELSuper SW Series; Tosoh Biosciences, Montgomeryville, Pa.), undernon-denaturing conditions. Proteins were identified using a micro flowcell UV detector at 280 nm. As shown in FIG. 2, a major protein speciesof 67.5 kDa was identified (retention time 18.747 minutes). The 67.5 kDapeak contributed about 90% of the total signal measured at 280 nm. Alsodetected were peaks at retention times of 21.544 minutes (21.0 kDa) and23.957 minutes. Analysis under denaturing and other conditions indicatesthat the 21.0 kDa species is a protein molecule and the 23.957 minutepeak is primarily polysaccharide. The major components of thephyto-percolate (the 67.5 kDa protein, 21.0 kDa protein, and thepolysaccharide identified at 23.957 minutes) are referred to herein asphyto-percolate derivatives and may contribute to the biological andtherapeutic efficacy of the phyto-percolate.

Another sample of the lyophilized phyto-percolate was subjected toFourier Transform Infrared (FTIR) spectroscopy. The results are providedin FIG. 3. FIG. 3 shows a spectrum that is characteristic of a dissolvedprotein sample.

A third sample of the lyophilized phyto-percolate was used for [¹H]-NMR.The NMR spectrum is provided in FIG. 4. Here again, the results areconsistent with a single protein species.

Weight Management Using Phyto-Percolate

Excessive weight has emerged as a prominent and growing health problem.Greater than 61% of Americans over the age of 20 are overweight, 25% ofwhom are obese. Second only to tobacco use as the top underlyingpreventable cause of death, excessive weight is a major risk factor fordeveloping diabetes, heart disease, hypertension, gallbladder disease,arthritis, lung diseases, and certain types of cancer.

EXAMPLE 1 Rodent Model of Weight Loss

A 21 day weight loss study using twelve mature (12 month old)Sprague-Dawley rats was performed. Each animal was orally administered10 ml/kg of undiluted and unfiltered phyto-percolate (i.e., rawphyto-percolate) for 14 days, followed by non-dosing for 7 days. Eachanimal was weighted daily and observed for signs of toxicity. As shownin more detail in Table 1, the rats lost an average of 33 grams (6.3%)of body weight over the initial 14 day dosing period. They immediatelybegan to regain lost body weight upon cessation of phyto-percolateadministration. By the 21 day time point (7 days of non-dosing), therats had lost an average of 25 grams (4.7%) of initial body weight(i.e., gained an average of 8 grams since phyto-percolate cessation).

The test animals were observed for adverse reactions immediately aftereach dose and at 4 and 24 hours subsequent. Daily observation foradverse reactions was continued during the 7 day non-dosing period.Specifically, clinical observations for adverse reactions were made forrespiration, motor activity, convulsions, reflexes, ocular signs,salivation, pilorection, analgesia, muscle tone, gastrointestinaleffects, and skin/dermal alterations. Gastrointestinal effects were theonly observed adverse reaction. Soft to loose stool was observed in alltest animals. No other adverse reaction was observed. TABLE 1 IndividualWeight Loss Data Pre-dosing 14 Day Weight Loss 21 Day Weight Loss TestWeight Weight (% Initial Weight (% Initial Subject (g) (g) Body Weight)(g) Body Weight) 1 484 443 41 (8.5%) 453 31 (6.4%) 2 482 461 21 (4.4%)479  3 (0.6%) 3 549 521 28 (5.1%) 531 18 (3.3%) 4 536 499 37 (6.9%) 50729 (5.4%) 5 510 462 48 (9.4%) 468 42 (8.2%) 6 488 459 29 (5.9%) 465 23(4.7%) 7 535 506 29 (5.4%) 514 21 (3.9%) 8 586 558 28 (4.8%) 562 24(4.1%) 9 569 504  65 (11.4%) 518 51 (9.0%) 10 522 492 30 (5.7%) 498 24(4.6%) 11 556 532 24 (4.3%) 537 19 (3.4%) 12 524 503 21 (4.0%) 507 17(3.2%) AVG 528.4 495.0 33.4 (6.3%)   503.3 25.1 (4.7%)  

EXAMPLE 2 Human Weight Loss and Glucose Control Study

A single-center, prospective, randomized, triple-masked,placebo-controlled parallel-group-design pilot clinical trial of thephyto-percolate was performed using two different batches of thephyto-percolate. This trial was conducted in accordance with FDAregulations and under a protocol approved by an Institutional ReviewBoard (IRB).

Subjects: Primary inclusion criteria were men and women having a bodymass index (BMI) of 25-40 m/kg², 18-70 years old (inclusive), anddesirous of losing weight. Major exclusion criteria were moderate tosevere co-morbid disease (e.g., cancer); history of stroke, transientischemic attack (TIA), or similar conditions; uncontrolled hypertension,insulin-dependent diabetes, renal disease, moderately severe cardiacdisease, lupus, alcohol abuse, and current or recent use of certainmedications including medications and/or supplements for weight loss,glucose management, or arthritis. Women were excluded if they werepregnant, nursing, or actively trying to become pregnant.

Protocol: Patients were assigned to self-administer one ounce offiltered phyto-percolate or placebo three times each day (t.i.d.) on anempty stomach at least 30 minutes before a meal. Subjects were asked toparticipate in a reduced carbohydrate diet and light exercise programand complete a one-day-per-week Food Log and a daily Exercise Log forthe duration of the clinical trial. Patients were evaluated during abaseline examination and then again at 2-week, 4-week, and 6-weekvisits. Evaluations included measurement of body weight, arm and waistcircumference, and body fat measurements.

Glucose Control Study: At the baseline examination and at the 4-week and6-week visits, patients' fasting (12 hour) blood glucose was measuredand then their blood glucose was measured one hour after a glucosechallenge (25 grams of jelly beans; 90.4% carbohydrate). The differencebetween the glucose challenge reading and the baseline reading in asingle visit is an indicator of the patient's ability to regulate serumglucose levels.

Test Materials: The patients in the treatment groups were assigned oneof two different lots (Batch 1 and Batch 2) of phyto-percolate preparedas described above. The placebo product was similar in appearance(color, viscosity, and odor) to the diluted phyto-percolate. All testmaterials were dispensed in unlabeled blue bottles with instructions torefrigerate after opening.

Enrollment: A total of 44 subjects were enrolled and randomized for thistrial. Ten subjects completed the study on Batch 1 (Cohort 1) of thephyto-percolate and twelve subjects completed Batch 2 (Cohort 2). Sevensubjects completed the placebo phase of the trial.

Results: There were no significant adverse events reported. Patients inthe treatment arms of the study reported greater energy and reducedhunger compared to the Placebo group. The remaining results are asfollows:

After 2, 4, and 6 weeks of treatment with the diluted filteredphyto-percolate, the average percent total weight loss (above placebo)for all treated patients (Cohorts 1 and 2; n=22) 77.7%, 48.5%, and68.1%, respectively. After six weeks of phyto-percolate treatment,Cohort 1 lost an average of 106% (9.03 lbs) and Cohort 2 lost an averageof 37% (6.01 lbs) more than the weight loss measured in the Placebogroup (4.39 lbs). TABLE 2 Average Weight Loss 2-Week 4-Week 6-WeekPlacebo 2.60 3.71 4.39 (n = 7) Cohort 1 5.71 6.81 9.03* (n = 10) Cohort2 3.71 4.43 6.01 (n = 12)p < 0.10 (unpaired Student's t-test)

TABLE 3 Frequency Distribution of Weight Loss in Individual Patients at6 Weeks Placebo Cohort 1 Weight Loss (number of patients) (number ofpatients) >+1 lb. — 1 +1 lb. > patient > −1 lb. — 1 −1 lb. > patient >−3 lb. 2 — −3 lb. > patient > −5 lb. 2 — −5 lb. > patient > −7 lb. 3 1−7 lb. > patient > −9 lb. — 3 −9 lb. > patient > −11 lb. — 2 −11 lb. >patient > −13 lb. — — −13 lb. > patient > −15 lb. — — −15 lb. >patient > −17 lb. — 1 −17 lb. > patient > −19 lb. — — <−19 lb. —   1 ** maximum weight loss was 28 lbs.

TABLE 4 Arm and Waist Circumference - Difference Between Baseline and 6Weeks Placebo Cohort 1 Cohort 2 Arm 0.083″ 0.41″ *  0.13″ Waist 1.09″2.08″ ** 1.34″* p < 0.042** p < 0.21

TABLE 5 Body Composition - Percent Body Fat: Difference Between Baselineand 6 Weeks Placebo Cohort 1 Cohort 2 Body Fat @ Baseline 39.1% 39.2%39.0% Improvement in Body Fat (lbs) 2.11 6.03* 2.89 Improvement in LeanMass (lbs) 0.16 0.79** 0.24*p < 0.01**p < 0.15

TABLE 6 Frequency Distribution of Body Fat Loss in Individual Patientsat 6 Weeks Placebo Cohort 1 Weight Loss (number of patients) (number ofpatients) >+1 lb. — 2 +1 lb. > patient > −1 lb. 2 1 −1 lb. > patient >−3 lb. 2 — −3 lb. > patient > −5 lb. 2 2 −5 lb. > patient > −7 lb. 1 2−7 lb. > patient > −9 lb. — — −9 lb. > patient > −11 lb. — 1 −11 lb. >patient > −13 lb. — — −13 lb. > patient > −15 lb. — — −15 lb. >patient > −17 lb. — 1 −17 lb. > patient > −19 lb. — — <−19 lb. — 1* maximum weight loss was 28 lbs.

TABLE 7 Serum Glucose Levels In Individual Patients In Cohort 1 (mg/dl)Pa- Baseline 4-Week 6-Week tient Fast Chal. Diff. Fast Chal. Diff. FastChal. Diff. 1 158 264 106 155 246 91 152 238 86 2 72 128 56 89 107 18 8094 14 3 75 135 60 87 130 43 91 117 26 4 73 128 55 78 74 −4 76 80 4 5 105151 46 104 127 23 103 125 22 6 139 210 71 129 198 69 126 181 55 7 145204 59 124 200 76 132 195 63 8 85 122 37 74 159 85 83 133 50 9 91 143 5291 125 34 92 121 29 10  78 119 41 92 99 7 88 98 10 Mean 58.3 44.2 35.9n >126* 3 2 2*values >126 mg/dl are indicative of diabetes.

TABLE 8 Group Mean Data For Glucose Tolerance Test (mg/dl) Baseline4-Week 6-Week Placebo 61.7 58.3 54.0 Improvement 3.4 (5.5%) 7.7 (12.3%)Cohort 1 58.3 44.2 35.9 Improvement 14.1 (24.2%) 22.4 (39.6%)* Cohort 260.6 56.2 55.4 Improvement 4.2 (6.9%) 5.2 (8.6%) *p < 0.08

Conclusions: The weight loss, improvement in body fat, improvement inglucose control, as well as energy and hunger categories over the courseof this six-week study for those on the phyto-percolate was strong,particularly when compared to the placebo group. Cohort 1 lost abouttwice as much weight (1.5 lbs/week) as the placebo group (0.78lbs/week). Seven of the ten subjects in Cohort 1 lost seven pounds ormore, while none of the seven in the placebo group lost that muchweight. Correspondingly, a significant reduction in waist size wasmeasured in Cohort 1.

Significant improvements also were measured in the glucose tolerancetest. Test subjects demonstrated an average of 2.6x (156%) and 1.7x(69%) improved glucose control at 4 weeks and 6 weeks, respectively,when compared to the placebo group. Furthermore, 6 of the 22 testsubjects met the clinically important criterion of >50% control overbaseline. Three of these six demonstrated complete control of theglucose challenge, defined as >85% glucose control over baseline.

In Vitro Anti-inflammatory Effects: COX-2 Inhibtion

Cyclooxygenase-2 (COX-2) is a key regulator of the inflammatory cascade.COX-2 inhibitors are believed to reduce inflammation by blockingprostaglandin production. In view of the adverse effects associated withmixed COX inhibitors (aspirin, ibuprofen, and naproxen) and thepresently available COX-2-specific inhibitors (valdecoxib, celecoxib,rofecoxib), there is a need for improved anti-inflammatory therapieswith fewer side effects.

Three separate preparations of the phyto-percolate were screened, usingan in vitro assay, for COX-2 inhibition. Riendeau et al., Can. J.Physiol. Pharmacol. 75: 1088-1095, 1997; Warner et al., Proc. Natl.Acad. Sci. USA 96: 7563-7568, 1999. Briefly, this assay measured toconversion of 0.3 μM arachidonic acid to PGE₂ by human recombinantinsect Sf21 cells expression human COX-2. The incubation buffercontained 100 mM Tris-HCl (pH 7.7), 1 mM glutathione, 1 μM hematin, and500 μM phenol. PGE₂ was quantified using an enzyme-linked immunoassay(EIA).

Sample 1 was a sample of diluted phyto-percolate concentratedapproximately 100-fold by drying under N₂. Sample 2 was prepared bydrying a 4800 μl sample of diluted phyto-percolate under N₂ andreconstituting it in 96 μl of ddH₂O just prior to assay. Sample 3 wasprepared by lyophilizing a 4800 μl sample of diluted phyto-percolate andreconstituting it in 96 μl of ddH₂O just prior to assay. Theconcentrations of phyto-percolate used, 100X, 10X, and 1X, refer to 10μl, 1 μl, and 0.1 μl of sample, respectively, in a final assay volume of100 μl. Rofecoxib was used as a positive control for COX-2 inhibition.Each sample was assayed in at least three concentrations and the assayswere performed in duplicate. TABLE 9 COX-2 Inhibition By Phyto-percolate% COX-2 Inhibition Sample Concentration (individual assay values) IC₅₀ 1100× 29 (27, 30.9) >100×  10× 11 (9.2, 13.4)  1× −4 (−9.0, 0.3) 2 100×61 (66.7, 56.1)  46.5×  10× 27 (23.7, 30.5)  1× 20 (13.3, 27.6) 3 100×58 (63.9, 52.3)  61.9×  10× 24 (21.7, 26.0)  1× 18 (13.3, 23.1)rofecoxib   1 μM 88 (90.1, 85.6) 0.198 μM  0.3 μM 55 (58.8, 51.8)  0.1μM 33 (34.7, 31.5) 0.03 μM 16 (22, 10.5) 0.01 μM 11 (8.2, 14)In Vivo Anti-inflammatory Effects: Carageenan-Induced Paw Edema

The carrageenan-induced paw edema assay was used as an in vivo indicatorof the anti-inflammatory effects of the phyto-percolate. Carrageenaninduces local inflammation and edema when injected into the paw pad of arat (Di Rosa et al., 1971). The development of paw edema is believed tobe biphasic (Vinegar et al., 1969). The initial phase is attributable tothe local release of histamine and serotonin (Crunkhon et al., 1971) andthe second phase is caused by prostaglandin release as a result of COXactivation. The second phase is measured as an increase in paw volumeand has been demonstrated to be responsive to steroidal andnon-steroidal anti-inflammatory agents.

Groups of test subjects (n=6) received oral doses of either vehiclecontrol (water; 5 ml/kg), indomethacin (30 mg/kg), aspirin (100 mg/kg),unfiltered phyto-percolate (10 ml/kg), or filtered phyto-percolate (10ml/kg) 30 minutes prior to intraplantar administration of carrageenan(0.1 ml of a 1% solution). Paw volume was measured at 0, 2, 4, 6, 8, and20 hours after treatment using a plesthysmometer to measure volumedisplacement. Each treatment group is compared to control.

As shown in Table 10, the paw volume of the control animals and alltreatment groups nearly doubled in two hours and remained so through thefour hour time point. By six hours, paw volume was reduced by 30% and50% in the groups administered the filtered and unfilteredphyto-percolate, respectively. This reduction in edema was significantlybetter than that observed for either the indomethacin or the aspiringroups at this time.

Further, the reduction in edema measured for the two phyto-percolategroups was comparable to both the indomethacin and aspirin groups at the8 hour and 20 hour time points. TABLE 10 In Vivo Anti-inflammatoryEffects of Phyto-percolate Mean paw volume (ml) ± SD (% change fromcontrol) Group 0 hours 2 hours 4 hours 6 hours 8 hours 20 hours Control1.24 ± 0.17 2.18 ± 0.24 2.17 ± 0.27 2.12 ± 0.15 2.05 ± 0.08 1.85 ± 0.08Indomethacin 1.25 ± 0.05 2.25 ± 0.23 2.18 ± 0.22 2.00 ± 0.22 1.83 ± 0.231.37 ± 0.10  (1%)  (7%)  (1%) (−12%) (−22%) (−38%) Aspirin 1.25 ± 0.082.22 ± 0.28 2.07 ± 0.23 1.92 ± 0.18 1.80 ± 0.18 1.42 ± 0.16  (1%)  (4%)(−10%)  (−20%) (−25%) (−23%) Filtered 1.22 ± 0.04 2.15 ± 0.10 2.15 ±0.10 1.78 ± 0.10 1.78 ± 0.10 1.35 ± 0.08 (−2%) (−3%) (−2%) (−34%) (−27%)(−30%) Unfiltered 1.20 ± 0.13 2.15 ± 0.12 2.13 ± 0.10 1.67 ± 0.10 1.67 ±0.10 1.28 ± 0.12 (−4%) (−3%) (−4%) (−45%) (−38%) (−37%)Immunological Effects: Rodent Model of HIV Infection

The effect of treatment using the phyto-percolate was investigated usinga rat model of HIV infection. The HIV model used inoculates rats withseven (7) of the nine (9) HIV genes, making it a non-contagious modelthat develops full symptoms of HIV by 9 months after inoculation, with alife expectancy of 12 months.

Some of the most devastating symptoms of HIV manifest themselves in theliver and the immune system. Liver problems are frequent causes ofillness and death in people with HIV infection. Throughout the study,liver function tests including AST, ALT, GGTP, bilirubin, and albuminwere monitored in the treatment and control groups. C-reactive proteinwas assayed as an inflammatory marker. The immune response was monitoredusing IgG, IgA, and IgM levels which are known to decline during theprogression of AIDS.

For testing, serum was drawn by cardiac puncture for baseline(pre-inoculation) values. The treatment group received dilutedphyto-percolate for their drinking water, which was allowed ad libitum,while the control group received filtered water. Serum was drawn bycardiac puncture, as above, every thirty (30) days until the terminationof the study.

After 60 days of treatment with the diluted phyto-percolate, thetreatment group had an average 30% increase in IgA levels, 50% increasein IgG levels, and a 40% reduction in C-reactive protein (C-RP) levels,relative to the untreated group (Table 11). No significant differencesin body weight, average daily food consumption, or average daily liquidconsumption were detected between the groups. TABLE 11 Serum AnalysisFrom Rat HIV Study Animal AST ALT Bilirubin C-RP IgG IgM IgA Group (U/L)(U/L) (mg/dL) (mg/ml) (mg/dL) (mg/dL) (mg/dL) Control Base 117 70 0.073.41 57 27 18 1 Mo. 95 60 0.12 0.65 69 26 24 2 Mo. 122 67 0.12 0.93 12026 24 HIV Base 116 77 0.07 3.37 60 26 21 1 Mo. 166 76 0.21 0.58 108 2725 2 Mo. 139 81 0.13 0.56 167 23 38Administration of Phyto-Percolate

The phyto-percolate dosage will vary with the severity of the disease,the biochemical activity of the disease, and the age and weight of thesubject. The effects of using the phyto-percolate will be measured usingstandard parameters known in the art for any such disease state.

In one embodiment, the phyto-percolate is orally administered as aliquid. As described in several of the foregoing examples, thephyto-percolate is diluted in filtered water to about 50 ppm of theprotein species of the 67.5 kDa peak measured by HPLC and UV detection(described above). However, depending upon the severity of disease ordesired clinical outcome, the concentration of phyto-percolate (andhence the dosage for the protein species) may be altered. For example,the protein species may be present in the orally administered liquid inconcentrations including about 100 ppm, 250 ppm, 500 ppm, 750 ppm, 1000ppm, 1500 ppm, or more. It is also contemplated that the proteinfraction is isolated from the phyto-percolate and formulated forparentera administration (e.g., intravenous, intramuscular, andsubcutaneous injection, topical, rectal or vaginal administration orother).

In an adult subject, the dosage of diluted phyto-percolate will varyfrom about one ounce per day, generally on an empty stomach, such as formaintenance and the retardation of aging, to about an ounce every hour,up to about 12 ounces per day, in a hospitalized burn or accident case,or during the chemotherapy infusion. The controlled diabetic orcardiovascular subject is generally treated at about two to three ouncesof phyto-percolate per day. Dosing on an empty stomach is noted becauseof the potential for interference on phyto-percolate function fromfood-stimulated gastrointestinal activities. A 50-70 lb. child is dosedat about three to four ounces per day, generally dosing on an emptystomach, during an acute infection. The greater the free radicaloxidative tissue destructive activity caused by age or disease state,the greater the recommended dosage of the phyto-percolate. Without beingbound to any particular theory, it is thought that the intake ofphyto-percolate per day is more directly related to the severity ofoxidative tissue destruction than to the weight of the subject.

It will be understood that various modifications may be made to theembodiments disclosed herein. Therefore, the above description shouldnot be construed as limiting, but merely as exemplification of thevarious embodiments. Those skilled in the art will envision othermodifications within the scope and spirit of the claims appended hereto.Likewise it should be understood that the phyto-percolate can be used toenhance the well being and performance of animals.

The foregoing has been a description of an illustrative embodiment ofthe present invention. While several illustrative details have been setforth, such are only for the purpose of explaining the presentinvention. Various other changes, omissions and additions in the formand detail thereof may be made therein without departing from the spiritand scope of the invention.

1. A method of treating or preventing a disorder in a mammal, saidmethod comprising administering to said mammal a therapeuticallyeffective amount of phyto-percolate or a derivative thereof.
 2. Themethod of claim 1, wherein said derivative is a protein having amolecular weight of about 67.5 kDa.
 3. The method of claim 1, whereinsaid derivative is a protein having a molecular weight of about 21.0kDa.
 4. The method of claim 1, wherein said derivative is apolysaccharide.
 5. The method of any of claims 1, wherein saidderivative has fibrinolytic enzymatic activity.
 6. The method of any ofclaims 1, wherein said phyto-percolate comprises between about 10 ppmand about 150 ppm of said derivative.
 7. The method of claim 6, whereinsaid therapeutically effective amount is between about 1 ounce to about12 ounces per day of said phyto-percolate.
 8. The method of any ofclaims 1, wherein said disorder is selected from the group consisting ofobesity, diabetes, inflammatory disorders, viral infections,cardiovascular diseases, cerebral vascular diseases, compromised immunesystem disorders, and metabolic disorders.
 9. The method of any ofclaims 1, wherein said disorder is obesity.
 10. The method of any ofclaims 1, wherein said disorder is diabetes.
 11. The method of any ofclaims 1, wherein said disorder is an inflammatory selected from thegroup arthritis, rheumatoid arthritis, ulcerative colitis, andinflammatory bowel disease.
 12. The method of any of claims 1, whereinsaid disorder is an HIV infection.
 13. The method of any of claims 1,wherein said disorder is gastric reflux disease.
 14. The method of anyof claims 1, wherein said mammal is selected from the group consistingof a human, a dog, a cat, and a horse.
 15. The method of any of claims1, wherein said compound functions as a broad spectrum anti-inflammatoryagent.
 16. A compound isolated from a phyto-percolate, wherein saidphyto-percolate is produced by culturing the microorganisms of ATCCDeposit # PTA-5863.
 17. The compound of claim 16, wherein said compoundis isolated from a culture of one or more of the microorganisms of ATCCDeposit # PTA-5863.
 18. The compound of claim 16, wherein said compoundis a protein.
 19. The compound of claim 16, wherein said protein has amolecular weight of about 67.5 kDa.
 20. The compound of claim 16,wherein said protein has a molecular weight of about 21.0 kDa.
 21. Thecompound of claim 16, wherein said compound is a polysaccharide.
 22. Apharmaceutical formulation comprising the compound of any of claims15-20 and a pharmaceutically acceptable excipient.